Figure 5.
STAT1 mediates 15(S)-HETE-induced CD36 promoter activity. A. CD36 promoter encompassing from −724 nt to +83 nt was cloned and sequenced. B. Left panel: Nuclear extracts of control and various time periods of 0.1 μM 15(S)-HETE-treated cells were analyzed by EMSA for STAT binding using STAT binding site at −107 nt as a [32P]-labeled probe. Right panel: Nuclear extracts of control and 2 hrs of 15(S)-HETE (0.1 μM)-treated cells were analyzed for the presence of STAT1 in the STAT-DNA complex by supershift EMSA. C. Upper panel: Control and various time periods of 0.1 μM 15(S)-HETE-treated cells were analyzed for STAT1 binding to CD36 promoter by ChIP assay. Middle & bottom panels: The levels of p47Phox, XO, Syk and Pyk2 were downregulated using their respective ASOs, quiesced, treated with vehicle or 0.1 μM 15(S)-HETE for 2 hrs and analyzed for STAT1 binding to CD36 promoter by ChIP assay. D. Upper panel: The CD36 promoter encompassing from −724 nt to +83 nt was cloned into pGL3 vector, transfected THP1 cells with the empty vector or pGL3-hCD36 promoter plasmid DNA, growth-arrested, treated with vehicle or 0.1 μM 15(S)-HETE for 6 hrs and the luciferase activities were measured. Middle panel: All the conditions were the same as in the upper panel except that after transfection and quiescence, cells were pretreated with Apocyanin (100 μM), DPI (10 μM), Allopurinol (100 μM), PF431396 (5 μM) or Bay61-3606 (10 μM) for 30 min before subjecting to treatment with vehicle or 0.1 μM 15(S)-HETE for 6 hrs and analyzing for luciferase activity. Bottom panel: THP1 cells were transfected with empty vector or pGL3-hCD36 promoter plasmid DNA with and without the mutated STAT-binding site (TTC was mutated to GCA), quiesced, treated with vehicle or 0.1 μM 15(S)-HETE for 6 hrs and luciferase activity was measured. *, p<0.01 versus vehicle control; **, p<0.01 versus 15(S)-HETE.